Mitochondrial Proliferation Comprising Polyglucan Nanofilaments

Glycosyltransferase MDR1 assembles a dividing ring for mitochondrial proliferation comprising polyglucan nanofilaments

Yamato Yoshidaa,1, Haruko Kuroiwab, Takashi Shimadac, Masaki Yoshidad, Mio Ohnumae, Takayuki Fujiwaraf, Yuuta Imotog, Fumi Yagisawah, Keiji Nishidai, Shunsuke Hirookaf, Osami Misumij,k, Yuko Mogia,l, Yoshihiko Akakabem, Kazunobu Matsushitam, and Tsuneyoshi Kuroiwab

aLaboratory for Single Cell Gene Dynamics, RIKEN Quantitative Biology Center, Osaka 565-0874, Japan; bDepartment of Chemical and Biological Science, Faculty of Science, Japan Women’s University, Tokyo 112-8681, Japan; cLeading Technology of Bioanalysis and Protein Chemistry, Shimadzu Corporation, Kyoto 604-8511, Japan; dIntegrative Environmental Sciences, Graduate School of Life and Environmental Sciences, University of Tsukuba, Ibaraki 305-8572, Japan; eNational Institute of Technology, Hiroshima College, Hiroshima 725-0231, Japan; fDepartment of Cell Genetics, National Institute of Genetics, Shizuoka 411-8540, Japan; gDepartment of Cell Biology, School of Medicine, Johns Hopkins University, Baltimore, MD 21205; hCenter for Research Advancement and Collaboration, University of the Ryukyus, Okinawa 903-0213 Japan; iGraduate School of Science, Technology and Innovation, Kobe University, Hyogo 657-8501, Japan; jDepartment of Biological Science and Chemistry, Faculty of Science, Yamaguchi University, Yamaguchi 753-8512, Japan; kDepartment of Biological Science and Chemistry, Graduate School of Sciences and Technology for Innovation, Yamaguchi University, Yamaguchi 753-8512, Japan; lDepartment of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan; and mDepartment of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753-8515, Japan

Edited by Krishna K. Niyogi, Howard Hughes Medical Institute, University of California, Berkeley, CA, and approved November 6, 2017 (received for review August 24, 2017) Mitochondria, which evolved from a free-living bacterial ancestor, membrane on the matrix side (Fig. S2D). Third, the dynamin contain their own genomes and genetic systems and are produced ring, which is composed of the mechanochemical GTPase from preexisting mitochondria by binary division. The mitochondrion- dynamin-related protein Dnm1, assembles as a ring-like struc- dividing (MD) ring is the main skeletal structure of the mitochondrial ture on the cytosolic side of the MD ring (Fig. S2E) (10–12).

division machinery. However, the assembly mechanism and molecu- Also, some additional mitochondrial division proteins have been PLANT BIOLOGY lar identity of the MD ring are unknown. Multi-omics analysis of identified (3–5), such as the WD40 repeat-containing protein isolated mitochondrial division machinery from the unicellular alga Mda1 (also known as “Mdv1” in yeast) (Fig. 1A) (13). Impor- Cyanidioschyzon merolae revealed an uncharacterized glycosyltrans- tantly, the MD ring appears to have the most distinctive structure ferase, MITOCHONDRION-DIVIDING RING1 (MDR1), which is specifi- among these rings; therefore, identifying the molecular compo- cally expressed during mitochondrial division and forms a single ring nents of the MD ring should provide insights into the funda- at the mitochondrial division site. Nanoscale imaging using immunoe- mental principles of mitochondrial division. To explore the lectron microscopy and componential analysis demonstrated that molecular components of the MD ring, we used the unicellular MDR1 is involved in MD ring formation and that the MD ring fila- red alga Cyanidioschyzon merolae (14, 15). C. merolae cells ments are composed of glycosylated MDR1 and polymeric glucose contain a single mitochondrion and a single chloroplast (plastid), nanofilaments. Down-regulation of MDR1 strongly interrupted mito- and the division of these organelles can be highly synchronized chondrial division and obstructed MD ring assembly. Taken together, (Fig. S1A). This alga is the only organism for which protocols our results suggest that MDR1 mediates the synthesis of polyglucan for isolating intact mitochondrial and plastid division machinery nanofilaments that assemble to form the MD ring. Given that a homolog of MDR1 performs similar functions in chloroplast division, the Significance establishment of MDR1 family proteins appears to have been a singular, crucial event for the emergence of endosymbiotic organelles. The mitochondrion-dividing (MD) ring mediates binary division of mitochondria. However, the molecular identity of the MD ring mitochondrial division | chloroplast division | endosymbiosis | is currently unknown. We show that the glycosyltransferase Cyanidioschyzon merolae MITOCHONDRION-DIVIDING RING1 (MDR1) regulates the synthesis of the polyglucan nanofilament bundle that assembles the itochondria are the descendants of an endosymbiosed MD ring. MDR1 is essential for mitochondrial division and forms Mbacterial ancestor. Due to their evolutionary origin, mi- a single ring at the mitochondrial division site in the unicellular tochondria are not synthesized de novo; instead, like free-living red alga Cyanidioschyzon merolae. Nanoscale imaging and bacteria, mitochondria proliferate via binary division of preex- componential analysis demonstrated that MDR1 is involved in isting mitochondria (1, 2). Mitochondrial division is controlled by MD ring formation and that the MD ring filaments are composed a ring-shaped supramolecular complex known as the “mito- of polymeric-glucose nanofilaments. An MDR1 homologue per- chondrial division machinery” (Fig. 1 A and B and Fig. S1)(1–5). forms a similar function in chloroplast division, suggesting that This chimeric structure includes three types of rings that origi- the establishment of the MDR1 family was crucial for the nated from bacterial and eukaryotic membrane fission systems: emergence of endosymbiotic organelles. the mitochondrion-dividing (MD) ring, the FtsZ ring, and the dynamin ring. The MD ring (6) is the main skeletal structure of Author contributions: Y.Y. and T.K. designed research; Y.Y., H.K., T.S., M.Y., Y.A., and K.M. performed research; Y.Y., H.K., M.O., T.F., Y.I., F.Y., K.N., S.H., O.M., and Y.M. ana- the machinery and is an electron-dense specialized structure that lyzed data; Y.Y., T.S., and M.Y. performed proteomic analyses; Y.A. and K.M. performed is occasionally observed as a fine filament bundle on the cytosolic componential analyses; and Y.Y., Y.M., and T.K. wrote the paper. side (Fig. 1C and Fig. S2 A–C) (7). However, previous studies The authors declare no conflict of interest. have indicated that the filaments of the MD ring are not com- This article is a PNAS Direct Submission. posed of conventional protein polymers such as actin and tubulin Published under the PNAS license. (2), and it is unknown which molecules constitute the MD ring. 1To whom correspondence should be addressed. Email: [email protected]. The FtsZ ring, which is composed of the bacterial division pro- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. tein homolog FtsZ (8, 9), forms beneath the inner mitochondrial 1073/pnas.1715008114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1715008114 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 A Saccharide staining E Interphase cell Dividing cell Conventional staining method method Isolated division machinery complex Mda1 mito CDPC mt

Mda1 Dnm2 B G1 S G2 M

n MD mt pt pt

PD G1 S G2 M Plastid division 5-repeat (kD) FHGIMito. division CMO005C Glycosyltransferase transmembrane J Dnm2 150- G1 S G2 M PDR1 domain domains 1000 Dnm1 Mda1/Mdv1 100- 54 FtsZ2-1 100 377 CMJ240C MDR1 70- (29%) FtsZ2-2 131 (700 a.a.) CMS045C 50- 800 Mda1 (71%) CMF051C CMT231C 3 37- Dnm2 CMQ054C 2 FtsZ1-2 25- CMT582C Annotated gene CMP225C bits 1 600 K E C Q GG L K P CMR014C I Q Uncharacterized gene 0 M S FI CMT564C F CC A L 0.5% NP-40 CMO170C CMT165C 211 212 213 214 215 216 IB: PS

Score CMT383C 217 218 219 400 CMJ262C/MDR1 K MDR1 CMP155C Mitochondrial division matrix PDR1 CMG179C marker CMQ199C G1 S G2 M G1 membrane CMQ208C marker 200 FtsZ2-1 CMR203C IB: 27 30 33 36 39 42 45 48 (hr) FtsZ2-2 Dnm1 MDR1 CMT274C MDR1 FtsZ1-2 CML145C M CMO131C 1234 CMK172C FtsZ1 IB: 0 CMO130C MDR1 CMD021C Mda1 0609030 120 150 180 −2 20 CMT566C Z CMQ200C control isolation Gene ID Row score CMF088C

Fig. 1. Identification and expression profiles of the mitochondrial division gene MDR1 via multi-omics analysis. (A) Phase contrast and immunofluorescence images of interphase (Left) and dividing (Right) cells of the unicellular red alga C. merolae. The mitochondrial division machinery and the mitochondria were immunostained with anti-Mda1 antibodies (Mda1, yellow) and antimitochondrial porin antibodies (mito, red). (Scale bar: 1 μm.) (B) Schematic representation of organelle- and cell-division processes of C. merolae. MD, mitochondrial division machinery; mt, mitochondrion; n, nucleus; PD, plastid division machinery; pt, plastid. (C and D) EM images of dividing C. merolae cells. The Inset in C shows the mitochondrial division site (boxed area in C). The MD ring (white arrowheads) was observed as electron-dense deposits on the top and bottom of a dividing mitochondrion (C) and was visualized by saccharide staining in early (D, Upper) and late mitochondrial division phases (D, Lower). (Scale bars: 200 nm in C and D; 100 nm in Inset.) (E) Immunofluorescence and EM images of isolated division machinery complexes containing mitochondrial division machinery (green) and plastid division machinery (red). The Inset shows a schematic representation of the mitochondrial (green) and plastid (red) division machinery in the EM image. (Scale bars: 500 nm, Left; 200 nm, Right.) (F and G) Mascot score histograms (F) and pie chart (G) of the 185 identified genes in the fraction of isolated division machinery complexes. (See Table S1.) (H, Left) Hierarchical clustering analysis of gene expression patterns of the identified genes using the time-course transcriptome dataset. (H, Right) The two groups that contain known endosymbiotic organelle division genes and exhibit specific expression patterns. (See Fig. S3.) (I) Domain architecture of CMJ262C/MDR1. A specific motif in the glycosyltransferase domain is visualized. (See Fig. S4.) (J and K) Immunoblot analysis of the protein expression profiles of MDR1 using anti- MDR1 antibody (J) in synchronized cell cultures (K). IB, immunoblot. (L and M) Comparison of the amounts of MDR1 protein at each isolation step. Twenty micrograms of protein per sample were loaded onto each lane. P, pellet; IB, immunoblot; S, supernatant. Lane 1, whole cell; lane 2, isolated mitochondria and plastid; lane 3, isolated mitochondrial and plastid membranes; lane 4, isolated division machinery complexes.

have been established (7, 16, 17). These biological features and involved in MD ring assembly using a multi-omics approach. We the availability of the complete genome sequence of C. merolae developed a protocol to isolate complex structures of the mito- have enabled the use of multi-omics approaches to study the chondrial and plastid division machinery (termed the “division underlying principles of mitochondrial and plastid division (Fig. machinery complex”)(Fig.1E and Fig. S1B) and subjected the S1 B and C). In this study, we show that the glycosyltransferase isolated fraction of the division machinery complexes to high- MITOCHONDRION-DIVIDING RING1 (MDR1) regulates resolution proteome analysis. Using this approach, we identified the synthesis of the polyglucan nanofilament bundle that assembles 185 genes, including 131 annotated genes (some of which were the MD ring. An MDR1 homolog performs a similar function in mitochondrial and plastid division genes) and 54 unknown genes plastid division, suggesting that the appearance of the MDR1 family (Fig. 1 F and G and Table S1). These genes, combined with a time- was a prime event for the establishment of endosymbiotic organelles. course transcriptome dataset from synchronized C. merolae cells (18), were classified into eight groups (groups 1–8) by hierarchical Results and Discussion clustering analysis (Fig. 1H, Left and Fig. S3). The genes in groups Identification of a Mitochondrial Division Protein, MDR1, Using a 2 and 6 showed specific gene-expression patterns indicative of a role Multi-Omics Analysis. To provide an initial molecular character- in mitochondrial division (Fig. 1H, Right and Fig. S3), including a ization of the MD ring, we analyzed C. merolae cells using a glycosyltransferase (CMJ262C) in group 6, which we designated combination of histochemical staining for saccharides and electron “MITOCHONDRION-DIVIDING RING1” (MDR1)(Fig.1I). The microscopy (EM). We identified positive electron-dense deposits primary feature of the deduced 700-aa sequence of MDR1 is a on the MD ring (Fig. 1D, arrowheads and Fig. S2 F and G), in- glycosyltransferase domain in its N-terminal region (residues dicating that saccharide molecules are a component of the MD 100–377) and putative five-repeat transmembrane (TM) do- ring. Thus, we next searched for the responsible glycosyltransferase mains in its C-terminal region (Fig. 1I). MDR1 homologs are

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1715008114 Yoshida et al. Downloaded by guest on September 24, 2021 widely conserved in various eukaryotes and possess a specific A Isolated division machinery complex signature in their glycosyltransferase domains (Fig. 1I and PC MDR1 PDR1 Merge Fig. S4). Immunoblot analyses using anti-MDR1 antibody (Fig. 1J) and synchronized cells showed that, like MDR1 mRNA expression (Fig. 1H), MDR1 protein expression was specifically detected during the mitochondrial division phase, as was ob- B served with the FtsZ1 and Mda1 proteins (Fig. 1K). After PC MDR1 Dnm1 FtsZ1 Merge nonionic detergent treatment of an isolated mitochondrial fraction using Nonidet P-40, MDR1 was retained in the insoluble fraction containing the division machinery complexes and Ring mitochondrial membranes and not in the soluble fraction con- PC MDR1 Dnm1 FtsZ1 Merge taining matrix components (Fig. 1L). Furthermore, we investigated the amount of MDR1 protein in the fractions obtained at each

isolation step by immunoblot analysis. When equal amounts of Spiral protein samples from each isolation step were subjected to im- Detergent-treated mitochondrial division machinery munoblot analysis, the concentration of MDR1 was found to in- C crease gradually with each isolation step and was most highly PC MDR1 Dnm1 FtsZ1 Merge concentrated in the isolated division machinery complex fraction (Fig. 1M). Taken together, these findings suggest that MDR1 is a mitochondrial division protein. PC MDR1 Mda1 FtsZ1 Merge Straight Intracellular and Subcellular Localization of MDR1 on the Mitochondrial Division Machinery. Immunofluorescence microscopy showed that fluorescence signals corresponding to MDR1 were not detected in the cell during interphase (Fig. 2A) and that MDR1 assembled into D E a single ring structure at the midregion of a mitochondrion just before the mitochondrial division phase (Fig. 2B). During mito-

chondrial division, the MDR1 ring was retained at the division site PLANT BIOLOGY (Fig. 2 C–F), indicating that MDR1 is an important component of the mitochondrial division machinery and is likely responsible for the contraction of the mitochondrial division site. Furthermore, MDR1 was uniquely and uniformly identified on the isolated mitochondrial division machinery (Fig. 3 A and B, Upper), even on the spiral form of the division machinery resulting from cutting off the ring-shaped machinery (Fig. 3B, Lower). To investigate whether MDR1 is an accessory or basal component of the MD ring, we examined the persistence of MDR1 proteins in the isolated mitochondrial division machinery via a partial solubilization assay using the zwitterionic detergent CHAPS. After the treatment, most of the isolated mitochondrial division machinery was present in straight form, and Dnm1, Mda1, and FtsZ1 were removed from the washed mitochondrial division machinery, but MDR1 protein was retained (Fig. 3C), suggesting that MDR1 is a fundamental component of the Fig. 3. Localization of MDR1 in the mitochondrial division machinery. (A) Protein localization of MDR1 (green) and PDR1 (red) in the isolated intact division machinery complex. (B) Protein distribution of MDR1 (green), Dnm1 PC MDR1 A mito D (red), and FtsZ1 (blue) in the isolated ring- and spiral-shaped mitochondrial division machinery. (C) Persistent protein localization of MDR1 on the mitochondrial division machinery after 2% CHAPS detergent treatment. (Up- per) Protein distribution of MDR1, Dnm1, and FtsZ1 on detergent-treated mitochondrial division machinery. (Lower) Protein distribution of MDR1, Mda1, and FtsZ1 on detergent-treated mitochondrial division machinery. B E (Scale bars in A–C:1μm.) (D) Immuno-EM micrograph of isolated mitochondrial division machinery. Immunogold signals (black particles) indicate protein localization of MDR1. (Scale bar: 100 nm.) (E) An enlarged micrograph of the boxed area in D. Red arrowheads indicate a single MD ring filament. (Scale bar: 25 nm.) C F MD ring. Based on these findings, we next explored the nanoscale localization of MDR1 in the MD ring by immunoelectron microscopy (immuno-EM) to identify the structural relationship between MDR1 and the MD ring. Immunosignals indicating MDR1 proteins were detected mainly in the inner periphery of the MD ring Fig. 2. (A–F) Phase contrast (PC) and immunofluorescence images of MDR1 (Fig. 3D). Furthermore, examination of a region of loosened filament (green) show intracellular localization of MDR1 throughout the cell cycle. The mitochondria were immunostained with antimitochondrial porin anti- bundles showed that immunogold particles localized to an MD ring bodies (mito, red). The Inset in B shows a ring structure formed by MDR1 nanofilament (Fig. 3E, arrowheads). These microscopy observations proteins from another sample. (Scale bar: 1 μm.) and the presence of putative TM domains in the MDR1 sequence

Yoshida et al. PNAS Early Edition | 3of6 Downloaded by guest on September 24, 2021 indicate that MDR1 localizes along the division site on the outer of C. merolae cells via treatment with the DNA synthesis inhibitor mitochondrial membrane and is incorporated into the inner periph- camptothecin (19), as the MD ring normally continues to mature ery region of the MD ring in vivo. at the mitochondrial division site in the cell (Fig. 4 A and B and Fig. S5). Thus we obtained overdeveloped mitochondrial division Identification of Glycosylated MDR1 and Glucose Molecules in the MD machinery, which appeared to have stronger immunofluorescence Ring Nanofilaments. To further clarify the molecular relationship signals from MDR1 (Fig. 4C) than the control (Fig. 3B). Immuno- between the MD ring, MDR1, and saccharide molecules, we ex- EM observation of the overdeveloped mitochondrial division ma- plored the componential profile of the MD ring. To increase the chinery showed that the MD ring consisted of a bundle of MD ring yield of isolated mitochondrial division machinery for subsequent filaments that was ∼1.8-fold thicker (bundle thickness ∼79.5 nm) analysis, we arrested both the cell cycle and mitochondrial division (Fig. 4D) than that of the control (bundle thickness ∼44 nm)

Mitochondrial division arrested cells Over-developed mitochondrial division machineries ABn C MDR1 PC MDR1 Dnm1 FtsZ1 Merge mito mito pt

Mda1

48 hr 60 hr Mitochondrial division machinery

+camptothecin D E

Control Mito. division Over-developed Arrest of cell-cycle and machinery Mito. division mitochondrial division Glycoprotein machinery staining

IB: MDR1

Over-developed mitochondrial division machinery

Mitochondrial division GHI25 Control Rha 20 Fru 15 Xyl G1 S G2 M Man 10 Glc 26 28 30 32 34 36 38 40 42 (hr) 5 MDR1 0 -5 FtsZ1-1 Mitochondria Row Z score 25 FtsZ1-2 2 20 ZED 15 Glc Mda1 0 10 Dnm1 Peak height (mV) 5 0 −2 UGP MDR1 MD ring -5 GS/SS 10 15 20 25 30 35 40 Retention time (min)

Fig. 4. Identification of the components of the MD ring filament. (A) Schematic of the isolation of overdeveloped mitochondrial division machinery from cell-cycle–arrested cells at the S–G2 phase. (B) Protein distribution of MDR1 in a cell-cycle–arrested cell produced by treatment with the DNA synthesis inhibitor camptothecin followed by synchronized cultivation for 48 and 60 h. (Scale bar: 1 μm.) (C) Phase contrast (PC) and immunofluorescence images of isolated overdeveloped mitochondrial division machinery. (Scale bar: 1 μm.) (Also see Fig. S5.) (D) Immunoelectron micrograph of isolated overdeveloped mitochondrial division machinery. Immunogold particles (10 nm in diameter) indicate the localization of MDR1 protein. (Scale bar: 100 nm.) (E) Comparison of fluorometric detection of glycoproteins in a 1D PAGE gel of the negative control, normal, and overdeveloped mitochondrial division machinery. Simulta- neously, immunoblot (IB) analysis of the same samples was performed with anti-MDR1 antibody. The negative control samples were prepared from insoluble fractions of isolated nondividing mitochondria and plastids after detergent treatment. (F) EM micrograph of the purified MD ring filaments from overdeveloped mitochondrial division machinery. (Scale bar: 200 nm.) (G) Component analysis of purified MD ring filaments by HPLC. HPLC profiles show that the filament fraction (Lower) contained only glucose (Glc) molecules. Fru, fructose; Man, mannose; Rha, rhamnose; Xyl, xylose. (H) Heatmaps of the gene-expression patterns of mitochondrial division genes and putative polyglucan synthesis-related genes such as UGP and GS/SS during the cell cycle in C. merolae. Gene-expression profiles of the genes were obtained using a dataset from a previous transcriptome analysis (18). (I) Schematic representation of the putative assembly process of MD ring filaments by MDR1.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1715008114 Yoshida et al. Downloaded by guest on September 24, 2021 (Fig. 3D). Using this fraction, we carried out an in-gel assay to MDR1 Plays a Critical Role in the Assembly of the MD Ring. Finally, detect glycosylated proteins to determine whether MDR1 is linked we knocked down MDR1 expression via antisense suppression with saccharide molecules. Specific signals from fluorescent- (SI Materials and Methods). Antisense-MDR1 significantly re- labeled glycosylated protein were detected in the isolated mito- duced the frequency of successful mitochondrial division com- chondrial division machinery fraction and the overdeveloped pared with control cells (P < 0.001; Fisher’s exact test) (Fig. 5 A division machinery fraction, whereas no signals were detected in and B), and immunofluorescence microscopy confirmed that a control sample derived from interphase cells (Fig. 4E, Upper). MDR1 was not expressed in the antisense-MDR1 cells (Fig. 5C), Immunoblot analysis confirmed that the signals were derived from suggesting that MDR1 is essential for mitochondrial division. In MDR1 (Fig. 4E, Lower). Moreover, the signal intensity of fluo- the antisense-MDR1 cells, FtsZ1 was expressed and localized to rescently labeled glycosylated protein was positively correlated with the mitochondrial division site, as in the control (Fig. 5D, Top that of MDR1 protein. Taken together, these results suggest that and Fig. S2D). However, fluorescence signals for Mda1 were not MDR1 binds to saccharide molecules. Next, we investigated which detected (Fig. 5D, Middle), and fluorescence signals for Dnm1 types of saccharide molecules bind to MDR1 and are included in were detected in the cytosol but did not accumulate at the mi- the MD ring filaments. After deproteinization of the isolated mitochondrial division site (Fig. 5D, Bottom). Previous studies have tochondrial division machinery (SI Materials and Methods), the MD shown that FtsZ proteins localize to the division site and form ring filaments were retained in the nondegradable fraction (Fig. the FtsZ ring before MD ring assembly (2, 11). After MD ring 4F). We analyzed the composition of this fraction and found that assembly, both Mda1 and Dnm1 proteins accumulate on the MD the MD ring filaments contained only glucose (Fig. 4G). Taken ring (11, 13). Therefore, the protein distribution patterns of together, these results indicate that the MD ring filaments are FtsZ1, Mda1, and Dnm1 in antisense-MDR1 cells were expected, composed of both MDR1 protein and polyglucan nanofilaments. and they reinforce the notion that MDR1 plays a critical role in Although the mechanism by which MDR1 synthesizes the MD ring the assembly of MD ring filaments and the formation of the MD filament is unclear, analysis of the glycosyltransferase domain of ring (Fig. S6 A and B). MDR1 indicated that it belongs to the glycosyltransferase-8 subfamily (Fig. S4A). As glycogenin, which catalyzes the biosynthesis of Appearance of the MDR1 Family Protein Might Be a Prime Event for glycogen from a UDP glucose donor substrate (20, 21), also belongs the Establishment of Endosymbiotic Organelles. The MDR1 ho- to this subfamily, we reasoned that MD ring synthesis may occur via molog PDR1 is required for the assembly of the plastid-dividing a mechanism that resembles glycogen synthesis. Indeed, the ex- (PD) ring, which consists of a polyglucan filament and performs

pression of UDP-glucose pyrophosphorylase (UGP), which catalyzes plastid division in C. merolae (17). Given that both mitochondria PLANT BIOLOGY the production of UDP-glucose (22), a central metabolite in car- and plastids evolved from free-living bacteria, the compelling bohydrate metabolism, increased just before the expression of structural and componential similarity between the MD and PD MDR1 (Fig. 4H). Glycogen/starch synthase (GS/SS)didnotshowa rings suggests that these ring structures were established in host phase-specific expression pattern (Fig. 4H), suggesting that the cells as endosymbiotic organelle-dividing (EOD) rings that synthesis and elongation of the MD ring filament might be medi- control the proliferation of endosymbiotic organelles during ated solely by MDR1 (Fig. 4I). early evolution (Fig. S7). When the earliest endosymbiotic event

A Control MDR1 antisense C mito mito PC MDR1 mito Merge pt pt anti. MDR1

D PC FtsZ1 mito Merge

Mda1 antisense MDR1 B Dnm1 Control (n = 124) MDR1 anti. * (n = 141) 035310 15 20 25 05 Successful rate of mitochondrial division (%)

Fig. 5. Knockdown of MDR1 by antisense suppression. (A) Phase contrast and fluorescence images of control and antisense-MDR1 cells. Mitochondria- targeted GFP-fused protein (mito, green) was used as a reporter protein. A plastid (pt) in the cells is visualized in red by autofluorescence. (Scale bar: 1 μm.) (B) Comparison of the rate of successful mitochondrial division (%) in control and antisense-MDR1 cells. *P < 0.001 as determined by Fisher’s exact test. Data are the total number of transformants examined (n) in more than five replicates. (C and D) Phase contrast (PC) and immunofluorescence images of MDR1 (C) and FtsZ, Mda1, and Dnm1 (D) in antisense-MDR1 cells. The mitochondria were immunostained with antimitochondrial porin antibodies (mito, red). (Scale bars: 1 μm.)

Yoshida et al. PNAS Early Edition | 5of6 Downloaded by guest on September 24, 2021 occurred in the host eukaryotic ancestor, the endosymbiont (re- Materials and Methods ferred to as “alpha-proteobacteria”) would not have divided and Synchronization and Isolation of Intact Mitochondrial and Plastid Division would have been transferred to one of the daughter cells during the Machinery. C. merolae strain 10D cells (14) were used in this study. Synchro- proliferation of the host eukaryote. Therefore, it is likely that the nization, isolation of mitochondria and plastids, and isolation of intact mi- host eukaryotic ancestor had to capture the endosymbiont re- tochondrial and plastid division machinery were performed as described peatedly via predation. Subsequently, a primitive division machinery previously (7, 17). See SI Materials and Methods for details. comprised of the MDR1-mediated EOD ring (the MD ring) emerged for proliferation of the endosymbiont, and this allowed the Liquid Chromatography-MALDI-TOF-MS Analysis, Data Analysis, and MASCOT Searches. The protein profiles of the samples were obtained by a MS/MS ions host cell to possess the endosymbiotic organelle permanently as search using a liquid chromatography (LC)-MALDI-TOF mass spectrometer in mitochondria via binary fission of preexisting mitochondria and reflectron mode. The LC-MALDI-TOF mass spectrometer comprised the fol- equipartition to daughter cells. Similarly, the plastid is also thought lowing: a Prominence nanoHPLC system (Shimadzu) and Presto FF-C18 column to have evolved from a cyanobacterial ancestor via the establish- (1.0 × 150 mm; Imtakt) for separating the peptide mixtures in the samples, ment of the division machinery comprised of the PDR1-mediated AccuSpot autospotter (Shimadzu) for spotting the separated samples onto the EOD ring (the PD ring). Concurrently, the appearance of EOD MALDI sample plates, and an AXIMA Performance mass spectrometer (Shi- rings (composed of specific glycosyltransferase proteins and poly- madzu) for MALDI-TOF/TOF-MS analysis. The identified proteins are listed in meric glucose nanofilaments) might have been a crucial, singular Table S1.SeeSI Materials and Methods for details. event for the emergence of endosymbiotic organelles. Although the molecular functions of MDR1- and PDR1-like Immunofluorescence Microscopy and Immunoblotting. Immunofluorescence microscopy and immunoblotting of whole cells and isolated mitochondrial and genes in higher eukaryotes remain enigmatic, these genes are plastid division machinery were performed as described previously (7, 17). The well conserved in various taxonomic groups of eukaryotes (Fig. primary antibodies used were anti-MDR1 (this study; see SI Materials and S4A). In addition, given the structural differences in domain ar- Methods), PDR1 (17), Mda1 (13), Dnm1 (11), FtsZ1 (11), mitochondrial elon- chitecture between MDR1 and PDR1, except for the presence of gation factor (EF)-Tu (19), and porin (7). See SI Materials and Methods the glycosyltransferase-8 domain (Fig. S4 A and B), other unchar- for details. acterized proteins containing the glycosyltransferase-8 domain might also function in a similar manner in mitochondrial and plastid EM. EM, immuno-EM, negative staining, and carbohydrate staining were per- division in higher organisms. Thus, further investigation of MDR1, formed as described previously (17). See SI Materials and Methods for details. PDR1, and the uncharacterized glycosyltransferase-8 proteins ACKNOWLEDGMENTS. This research was supported by Human Frontier Sci- should provide insights into long-standing questions about the ori- ence Program Long-Term Fellowship LT000356/2011-L (to Y.Y.) and a Japan gin of eukaryotic cells and the conserved proliferation mechanisms Society for the Promotion of Science Postdoctoral Research Fellowship for of mitochondria and plastids throughout eukaryotic cells. Research Abroad (to Y.Y.).

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